Alginates (FMC Biopolymer) and agarose were covalently coupled with the integrin binding peptide (Gly)4-Arg-Gly-Asp-Ala-Ser-Ser-Lys-Tyr (Peptides International)17 (link),20 (link). PEGDM polymers were photo-crosslinked in the presence of acryloyl-PEG-GRGDS21 (link). For cell encapsulation studies, mixtures of varying wt % polymer were mixed with stem cells (20 million clonally derived mMSC (D1) per mL, or 15 million hMSC/mL) and crosslinked to form hydrogels. The elastic modulus E of hydrogel matrices was measured using an Instron 3342 mechanical apparatus at a compression rate of 1mm/min. Cell-encapsulating hydrogels were transferred to FBS-supplemented Dulbecco’s Modified Eagle Media (DMEM, Invitrogen) containing a combination of osteogenic and adipogenic chemical supplements. After 1 week in culture, lineage specification was assessed by in-situ staining for Alkaline Phosphatase Activity (Fast Blue) and Neutral Lipids (Oil Red O) in the same samples, by OCN staining in cryosectioned matrices, or by biochemical analysis of cell lysates obtained by recovering cells from alginate matrices with 50mM ethylenediaminetetraacetic acid (EDTA) in PBS. Western analysis of cell phenotype was performed to assess population-level expression levels of FN and ColI, as well as adipogenic (PPAR-γ, Adn) and osteogenic (Cbfa-1, Osteopontin) biomarkers.
In certain experiments, cell, encapsulating, calcium-crosslinked alginate matrices were combined with cell-free alginate matrices in the same media so that the concentration of calcium ions available to cells could be controlled independent from the rigidity of cell-encapsulating matrices (Fig. S3). The relative diffusion coefficient for a model protein, bovine serum albumin (BSA; 67 kDa) was measured via release of rhodamine-labeled BSA from alginate matrices of varying rigidity (Fig. S3).